SUMMARY There remain major gaps in understanding regulation of human intestinal electrolyte transport under normal physiologic conditions and in diarrheal diseases. This proposal deals with the role of the brush border (BB) Cl/HCO3 antiporter, SLC26A3 (DRA) in intestinal Na, Cl and HCO3 transport and will resolve the gap in understanding of its acute regulation, intestinal cells involved and ways to reverse the abnormal transport in severe, cAMP related diarrheas. The long term goal of this project is to define the coordinated and dynamic changes in Na, Cl and HCO3 transport that occur in diarrhea so that they can be targeted by drugs to reverse the changes and treat most diarrheal diseases. The Aims of this proposal include to: I.Test the hypothesis that necessary steps in cAMP/cholera toxin inhibition of neutral Na and Cl absorption that involves DRA, which occurs at the same time as stimulation of DRA activity and the related HCO3 secretion, includes increased trafficking of DRA to the BB and reduction in the physical association of DRA with NHE3 that occurs under basal conditions and increased physical association with CFTR. II. Test the hypothesis that cAMP related changes in DRA and NHE3 activities and trafficking involve association with SNX27 in early endosomes and these changes can be reversed by retromer stabilizers, which identify a new drug target to treat diarrhea. To accomplish these studies we will use ileal and proximal colonic enteroids made from healthy human subjects grown as 2D monolayers and compare the results with those from the human colon cancer cell line, Caco-2 cells. Caco-2 cells are used for many studies of intestinal physiology and for drug development, including anti- diarrheal drugs. We predict that Caco-2 cells will give similar information regarding intestinal Na, Cl and HCO3 transport under basal conditions and in models of diarrhea; however it will be important to identify any differences that could be relevant to studies of intestinal function and drug development. These studies are innovative being among the first to study electrolyte transport in human enteroids as monolayers, to examine the interaction of DRA with NHE3 and CFTR in the same cell population with emphasis on their dynamic interactions with each other under basal conditions and with elevated cAMP, are the initial study of the role of the early endosomal protein SNX27 and the retromer in acute DRA regulation, and develop studies of SNX27/retromer as a potential drug target for future development to treat diarrhea. The expected outcomes of this proposal will be to a) understand the coordinated regulation of DRA, NHE3, and CFTR in a model of severe diarrhea that includes consideration of mechanism of HCO3 loss, a gap in understanding the pathophysiology of diarrhea, b) establish that drug therapy of diarrhea must consider the coordinated regulation of DRA with NHE3 and CFTR and not just the regulation of each transporter alone, and c) identify a new drug target in a diarrhea model in human intestine that can reverse the diarrhea related transport changes in DRA and NHE3, and that potentially could be developed into a drug to treat diarrhea.